Direct flow injection analysis nebulization electrospray and APCI mass spectrometry

ABSTRACT

A method and apparatus for Flow Injection Analysis (FIA) into Atmospheric Pressure Ion sources (API) including Electrospray (ES) and Atmospheric Pressure Chemical Ionization (APCI) sources whereby the sampling and spray needles are one and the same. The sampling and spray needle configured with an autoinjector apparatus or used in manual injection is introduced directly into a mating ES or APCI probe configured in an API source. Such a sampling and spray needle eliminates the need for injector valves, transfer lines or additional fluid delivery systems in FIA into API sources interfaced to mass spectrometers or other chemical analyzers. The use of a sampling and spray needle configuration reduces component costs, liquid dead volume, sample dilution effects, and minimizes cross contamination effects, solvent consumption and waste while increasing sample throughput.

RELATED APPLICATIONS

The present application is a continuation of application Ser. No.09/532,724 filed Mar. 22, 2000, now U.S. Pat. No. 6,458,597, whichclaims all rights of priority to U.S. Provisional Application Ser. No.60/125,492 filed Mar. 22, 1999, the contents of which are fullyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to apparatus and methods in the field ofmass spectrometry.

BACKGROUND OF THE INVENTION

Flow injection analysis (FIA) performed with an atmospheric pressure ion(API) source interfaced to a mass spectrometer (MS) is a common methodfor introducing sample into an API MS apparatus. Typically, API MS FIAis performed by connecting a sample injector valve in-line between asolvent or solution delivery system and an inlet probe to an API source.When the injector valve is switched to the load position, samplesolution is loaded into the injector valve through an injector needlewhile the separate solvent delivery system provides liquid flow to theAPI source probe through a separate channel in the injector valve. Theinjector valve is then switched so that the loaded sample solution,usually contained in a tubing loop or channel, is connected to thesolvent delivery channel and the sample solution flows into the APIsource through the API probe at a flow rate set by the solvent deliverysystem. The injector valve is generally connected to the API sourceprobe with a transfer line or tube. The sample is introduced into theinjector valve through an injector needle that is connected to a fluidreservoir or transfer line. The injector needle may be connected to asyringe for manual injection, a syringe configured in an autoinjector ortransfer tube or line that is connected to a remote fluid delivery meansconfigured in an autoinjector. In some commercially availableautoinjectors, the injector needle is attached directly to a syringe.The syringe and injector needle are loaded, emptied and positioned bythe autoinjector apparatus. The autoinjector moves the injector needleinto a vial or container holding sample solution and loads a programmedvolume of sample solution into the injector needle and attached syringe.The autoinjector apparatus then moves the syringe and injector needleposition to the injector valve and loads the sample solution into theinjector valve. To avoid sample carryover from one sample solutioninjection to the next, the injection needle inner bore and outer surfaceand the syringe inner volume may be washed or flushed between sampleinjections.

Other commercially available autoinjectors do not attach the injectorneedle directly to a syringe but instead connect the injector needle toa fluid transfer line or tube that is in turn connected to a syringe orfluid pump which may or may not translate with the injector needleposition. Sample solutions are drawn into the injector needle andconnected tubing and injected into the injector valve by activating theremote syringe or fluid delivery pump when the injector needle isappropriately positioned in a sample vial or the injector valverespectively. A number of apparatus and methods have been employed incommercial autoinjectors to flush or wash the outer and inner bore ofinjection needles and the connected tubing between sample injections.Typically, autoinjector needles are metal tubes with sufficient rigidityto push through the seal of an injector valve or sample vial top. Theflow rate of sample solution pulled into or delivered from the injectionneedle is programmably controlled by the autoinjector syringe orpositive displacement fluid flow pump. Autoinjectors can be programmedto inject sample solutions drawn from multiple sample vials orcontainers in an unattended sequence. Each sample loaded into theinjector valve is subsequently injected or delivered to the API MS wherea portion of the sample is ionized and mass to charge analysis.Alternatively, some or all sample solution transfer, injection andinjector needle cleaning steps performed by an autoinjector can beperformed manually as well with a handheld syringe or a syringe mountedon an syringe pump that is connected through a transfer line and aninjection needle to the injector valve.

API MS performance can be reduced using conventional FIA configured withinjector valves and transfer lines. MS signal resulting from ES and APCIsource ionization is essentially sample concentration dependent.Dilution of the sample can occur in injector valves and transfer linesdue to diffusion of sample solution into the mobile solvent, mixingconnection points and in dead volumes and adsorption to the walls. Suchdilution can result in reduced MS signal or tailing of injection peaks.Sample that has adsorbed to surfaces in the injector valve or liquidtransfer lines can bleed off during subsequent sample injections. Suchsample carry over can appear as added peaks or chemical noise insubsequent injections and may cause errors in trace component orquantitative MS analysis. The effects of the dead volumes from injectionvalves, connections and fluid deliver or transfer lines becomeincreasingly pronounced as the liquid flow rate or sample concentrationdecreases. When the liquid flow rate is decreased, the sample transittime in the injector valve and transfer tubing increases for a givendead volume. Longer sample transit times allow increased samplediffusion into the solvent, diluting the sample. Higher liquid flowrates may require more total sample to be injected to accommodate slowerMS data acquisition rates encountered with scanning mass spectrometerssuch as quadrupoles.

The Electrospray needle in some commercial ES sources is operated atkilovolt potentials during spraying. For such ES sources, a longerdielectric liquid transfer line of several inches is typicallyconfigured between the ground potential injector valve and the ES needleto allow a gradual drop in kilovolt potential through the samplesolution. A high electric field gradient in the transfer tube is avoidedto minimize sample heating, electrophoretic and electrolysis effectsduring FIA. Liquid transfer lines can be reduced in length when an ESsource in configured with a grounded needle, however, even with groundedES needles, the dead volume due to the transfer lines cannot be entirelyeliminated. For API MS FIA applications where small amounts of sampleare available for injection, sample dilution or losses due to injectorvalve, connector and transfer line dead volumes and surfaces maycompromise the limit of detection. Sample handling techniques employedin conventional FIA apparatus and methods may be the primary limitationin achieving lower limits of detection in API MS FIA analysis.

The invention reduces or eliminates those elements configured and usedin conventional FIA apparatus and methods that reduce API MS FIAperformance. In one preferred embodiment of the invention, the injectorneedle and an ES source has been configured such that the samplesolution can be sprayed directly from the injector needle tip. Theinjector needle tip is introduced into the ES source chamber through aprobe that serves as a needle guide, seal, electrical connection andpneumatic nebulization second needle layer. The injector needle can beintroduced into an APCI source through a similar probe apparatus servingas a needle guide, seal and pneumatic nebulization sprayer second tubelayer. Multiple injection needles can be configured to spray in amultiplexed manner through one or more API probes to increase FIA samplethroughput. The injector needle can be configured as a reusable ordisposable tip. The liquid spray flow rate is set by the auto or manualinjector sample injection flow rate. This flow rate can be set tooptimize MS analysis and sample throughput. The invention reducesinstrument cost by eliminating the need for an injector valve andcontrols, transfer lines and a separate solvent flow pump in API TOFFIA. The invention also minimizes solvent consumption and waste.

The invention allows increased sample throughput in API MS FIAapplications by eliminating steps and the time associated with liquidtransfer per injector needle. In one embodiment of the invention,multiple injector needles can be sequentially introduced into one APIsource probe or multiple injector needles can be introduced into an APIsource through multiple API source probes. T. Wang et. al., Proceedingsof the 46^(th) ASMS Conference on Mass Spectrometry and Allied Topics,1034, 1998 have reported the configuration and use of multiple injectorneedles and valves to shorten analysis run time and increase samplethroughput. Commercially available autoinjectors, such as the GilsonMultiprobe 215 liquid handler, have been configured with up to eightautoinjector needles dispensing to eight autoinjector valves whichtransfer sample through an additional selector valve to an API source.Fluid flow through such a system is provided by a separate liquid flowpump. The transfer lines have increased length from multiple injectorvalves when compared to the single injector valve configuration. Theincreased transfer and dead volumes from each injector valve through thetransfer lines and the switching valve to the API source must bethoroughly flushed between injections. The speed of injections even withsuch a multiple injector valve configuration is still limited to someextent by the washing and flushing of the eight injector valves,transfer lines and switching valve. In one embodiment of the invention,multiple injector needles can be configured for introduction into one ormore API probes without the need to add multiple injector valves,transfer lines, switching valves or an additional fluid flow pump.Increases in sample throughput can be achieved with the invention at alower cost, when compared with commercially available systems, without areduction in performance that is unavoidable in API MS FIA apparatuswith higher dead volumes. In the invention internal flushing or cleaningis limited to the injector needle and the attached reservoir andexternal flushing is limited to the injector needle only to avoid crosstalk or contamination sample carry over from one injection to another.Flushing or cleaning of valves or transfer lines is eliminated in FIAaccording to the invention.

SUMMARY OF THE INVENTION

The invention comprises a reusable or disposable injector needleconfigured in an autoinjector or a manual injector which serves as themeans to remove a sample solution from a container and transport suchsolution to an API source wherein the injector needle, when introducedinto the API source, serves as the spray needle to deliver sampledirectly into the API source chamber. Such fixed or disposable injectorneedle, when introduced into an API source, becomes the liquidintroduction channel or tube in the nebulizer probe of an APCI source,the nebulizer apparatus of a pneumatically assisted Electrospray probeor an Electrospray tip in an unassisted ES ion source probe. Ionsproduced from samples introduced through such sprayers into an APIsource are subsequently directed into vacuum where they are mass tocharge analyzed. Ions transported into vacuum from such API sourceapparatus may also be subject to mass to charge selection and/orfragmentation in MS/MS or MS/MS^(n) analysis. An API source may beconfigured with multiple direct injection needles and/or probes forintroducing samples at an increased rate into an API source.Autoinjectors may be configured with multiple injector needlesconfigured for direct delivery of sample into an API source through oneor more probes. Such multiple needle autoinjectors may deliver samplesin a sequential or multiplexed manner to such single or multiple directinjection API source ports or probes to maximize sample throughput. Inone embodiment of the invention, a reusable or disposable sampling andspray injection needle may be packed with material, such as C18 coatedbeads, to aid in desalting, sample cleanup or the separation of samplecompounds in solution during the sample pickup, delivery and spraysteps. Different solvent composition layers can be pulled sequentiallyinto such packed sampling and spray needles with attached reservoirsprior to sample pickup. The sample can then be sprayed into an APIsource from such a loaded injection needle using solvent gradients toaid in sample desalting, additional cleanup or sample compoundseparation during spraying.

Washing or flushing of a packed or open disposable injection needle,according to the invention, is not required between injections allowingan increase in sample throughput. In one embodiment of the invention,sample solution may be drawn up into a packed or open disposableinjection needle. The injection needle is subsequently introduced intothe API source and sample solution is sprayed from the injection needletip with or without a solvent gradient to elute sample from any packedmaterial. Alternately, a sample solution can be loaded into anon-disposable or reusable needle and the needle is then inserted intoand forms a seal with a packed disposable injection needle. The packedinjection needle is then introduced into an API source and the samplesolution and any solvent gradient flows from the non-disposable needlethrough the packed disposable needle. The resulting solvent and samplesolution is sprayed from the disposable needle tip into an API source.The packing material in the disposable tip serves to desalt or furtherclean the sample solution as well as to provide some sample componentseparation due to solvent gradient flow, if desired. Depending on therequirements of a specific analytical application, packing material maybe replaced by filter media according to the invention to aid in samplecleanup with a minimum of dead volume.

The invention eliminates the need for sample injector valves or transferlines into an API source, reducing sample dilution, loss andcontamination due to sample handing and transfer. When a reusable needleis configured in the invention, the needle inner bore and outer surfacecan be washed in between each sample delivery and spraying step toreduce or eliminate, chemical noise, cross talk or carry over from onesample to the next. The use of disposable needles, configured accordingto the invention, eliminates sample to sample cross talk orcontamination without a wash step between sample injections into the APIsource. Faster cycle times or more rapid sample injection throughput canbe achieved by eliminating wash steps. Alternatively, a wash step can berun for one or more reusable injection needles while sample delivery andspraying is occurring with another injection needle or needles. Theinvention reduces apparatus costs, sample losses, sample contamination,and sample handling and minimizes solvent consumption and waste whileincreasing sample throughput in flow injection analysis with Atmosphericpressure ion sources. A direct injection needle apparatus may beconfigured with other API inlets in the same API source chamber as ameans to increase analytical flexibility within one API sourceapparatus. Ions produced from the API source may be analyzed byapparatus other than MS including but not limited to ion mobilityanalyzers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of a sampling and spray injection needle that isintroduced into an ES source through an ES probe assembly whose axis isconfigured at an angle orthogonal to the axis of the vacuum entranceorifice.

FIG. 2 is a diagram of the progression of sample loading and sprayingwith a reusable injection needle attached to a syringe reservoir in FIAaccording to the invention.

FIG. 3 includes two diagrams of reusable injector needles inserted intoAPI probe assemblies with two different gas seals. The reusable injectorneedles are configured with syringes.

FIG. 4 is a diagram of a sampling and spray injection needle that isintroduced into an ES source through an ES probe assembly whose axis isconfigured at an angle substantially the same as the axis to the vacuumentrance orifice.

FIG. 5 is a diagram of a sampling and spray injection needle that isintroduced into an APCI source through an APCI pneumatic nebulizer probewhose axis is configured at an angle orthogonal to the axis of thevacuum entrance orifice.

FIG. 6 is a diagram of a sampling and spray needle that is introducedinto an APCI source through an APCI pneumatic nebulizer probe assemblywhose axis is configured substantially the aligned with the axis of theorifice into vacuum.

FIG. 7 is a diagram of a reusable injector needle configured with asyringe wherein the injector needle is filled with material to removecontaminants and separate sample components during FIA. Operation withsolvent gradients in the syringe is illustrated.

FIG. 8 is a diagram of removable injector needles configured with andwithout conductive elements attached and configured with and withoutfilled tips.

FIG. 9 is a diagram of the progression of sample loading and sprayingwith removable injector needles into an API source through an API sourceprobe assembly according to the invention.

FIG. 10 is a diagram syringe and pipette devices attached to differentconfigurations of removable injector needles.

FIG. 11 includes a Total Ion Chromatogram and five representative massspectra acquired in accordance with the invention by repeated injectionsof different samples using an autoinjector configured with a samplingand spray injector needle that is introduced into an ES sourceinterfaced to a mass spectrometer.

DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED EMBODIMENTS

The apparatus and methods used for flow injection analysis typicallyinclude an injector valve, transfer lines, fluid line connections, anaddition fluid delivery pump, a sprayer probe with internal volume forES and APCI sources and a switching valve when multiple injector valvesare configured. Each of these elements adds to the dead volume or mixingvolume encountered when delivering a sample solution into an API sourcein flow injection analysis. Added dead or mixing volumes can causesample dilution due to diffusion or mixing of the sample with solventduring sample solution flow into an API source. Sample can adsorb to thewalls of the valve, transfer line and probe transfer tube. Dilution ofsample and loss of sample to the inner surfaces of the flow pathwayresults in reduced ion signal and analytical sensitivity. As liquid flowrates are reduced the sample solution spends more time in the transferdead volumes. Increased transfer time results in increased sampledilution and loss to transfer surfaces. Adsorbed sample can bleed offvalve, transfer line, connector and probe surfaces in subsequentinjections, contributing chemical noise and interference peaks toacquired mass spectrum. Chemical noise or interference peaks due tocontamination from prior injected sample can reduce the accuracy ofquantitative measurements and compromise the limits of detection.Increased valve, connector, transfer line and probe surfaces requireincreased solvent flushing or cleaning time in between sample injectionsto minimize subsequent sample carry over or bleed. This requiredflushing increases solvent consumption and increases the time betweeninjections. Increased cleaning time between injections decreases thenumber of samples that can be injected in a given time period, reducingsample throughput.

The invention allows rapid flow injection analysis over a wide range ofliquid flow rates while minimizing solvent consumption and waste andeliminating all injector valves, fluid line connectors, transfer lines,probe liquid transfer tubes and additional liquid flow delivery systemapparatus. Sample dilution or adsorption losses and solvent consumptionare minimized with the invention and apparatus costs are reduced byelimination of components. Sample carry over or cross talk can beminimized with washing of reusable injection needles or eliminated withdisposable or removable injection needles configured according to theinvention. The invention comprises the configuration and use of aninjector needle to draw up sample solution from a sample vial orcontainer into the injector needle and attached solvent reservoir,transfer of the sample solution to an API source probe, passing of theinjector needle through the API source probe channel and spraying of thesample solution from the tip of the injector needle into an API source.Ions are produced from the sprayed solution in the API source and aredirected into vacuum where they are mass to charge analyzed.Alternatively, the ion population produced in the API source can be massselected and fragmented in MS/MS or MS/MS^(n) analysis. API sources mayinclude but are not limited to ES, APCI or Inductively Coupled Plasma(ICP) ion sources. Mass to charge analysis can be conducted by any typeof mass spectrometer including but not limited to quadrupoles, triplequadrupoles, Time-Of-Flight, three dimensional ion traps, FourierTransform Mass Spectrometers (FTMS) or magnetic sector massspectrometers.

One embodiment of the invention is diagrammed in FIGS. 1 and 2 in whichreusable injection needle 100 connected to syringe solvent reservoir 108is configured to serve as an Electrospray needle in ES source 105. FIG.2 is a diagram of the uptake of a sample solution from a sample vial orcontainer, transfer of said sample solution to an ES source probe andthe spraying of such sample solution into the ES source chamber usingthe same injector needle with attached solvent reservoir. Referring toFIG. 2, reusable injector needle 100 and attached syringe 108 are usedto draw up sample solution 122 from sample vial or container 123 indiagrams A and B. In diagram A, syringe plunger 120 with plunger seal121 is located in the full forward position in syringe 108. The insidebore of syringe 108 and injector needle 100 have been flushed withsolvent to remove any previously injected sample. The outside surface ofinjector needle 100 has also been rinsed with solvent to remove anypreviously injected sample. With plunger 120 moved to the full forwardposition, cleaned injector needle 100 with tip 113 is inserted intosample solution 122 in sample vial or container 123. Plunger 120 isretracted or moved in the reverse direction to pull sample solution fromsample vial 123 through the bore of injector needle 100 and into syringevolume 124 as diagrammed in FIG. 2B. Syringe 108 loaded with samplesolution is moved from the sample vial position as shown in diagram C toa position where injection needle 100 is in-line with bore 130 of ESprobe assembly 101 as shown in FIG. 1. Injector needle 100 is then movedthrough bore 130 of ES probe assembly 101 passing through entrance port111, seal 127, tube 106 and tip 102. Electrical connection withelectrically conductive injector needle 100 and ground or voltage supply131 is made through contact 125 attached to injector needle 100.

Kilovolt electrical potentials are applied to cylindrical lens 115,nosepiece 112 and the capillary entrance lens 114 in ES source 105 whenthe injection needle and ES probe assembly 101 are operated at ground orzero electrical potential. Nebulization gas 128 is introduced throughgas connection 107 and flows through the annulus described by the innerbore of tube 106 and the outer surface of injector needle 100 exiting attip 102. Plunger 120 is moved forward causing sample solution to exitthe syringe at injector needle tip 113. The sample solution isElectrosprayed with pneumatic nebulization assist from tip 113 due tothe local electrical field gradient at tip 113 and the exitingnebulizing gas 128. Spray 126 comprises charged liquid droplets thatevaporate as they move through ES chamber 103. A portion of theevaporating charged liquid droplets are directed by the electric fieldto move against counter current drying gas flow 110 toward capillaryentrance orifice 109. Ions are released from such evaporating chargeddroplets and are directed into vacuum through capillary 104. Ionsentering vacuum are subsequently mass to charge analyzed with a massspectrometer. Details of the counter current drying gas 110 andcapillary 104 configuration and function in API sources in described inU.S. Pat. Nos. 4,531,056 and 4,542,293 respectively and incorporatedherein by reference. The liquid flow rate of spray 126 is controlled bythe forward movement rate of syringe plunger 120. The filling, emptyingand positioning of syringe 108 with injector needle 100 may be manuallycontrolled or mechanically controlled as part of a programmableautoinjector or automated sample handling system to achieve API MS FIA.

Commercially available autoinjectors such as the Leap HTS PAL system areconfigured with syringes for the uptake, movement and injection ofsamples into injector valves. The syringes and attached injector needlesare typically mounted to a programmable x-y-z position translator arm.Under pre-programmed control, sample solution is removed from a selectedsample vial or vials, the loaded injector needle is moved to a positiondirectly in-line with the bore of an ES probe assembly and the injectorneedle is introduced through the bore of the ES probe assembly in an ESsource as diagrammed in FIGS. 1 and 2. Some commercially availableautoinjectors are configured with multiple syringes. FIA samplethroughput can be increased according to the invention when suchmultiple syringe autoinjectors are used. Such a multiple syringeautoinjector configuration can be operated whereby one syringe isspraying sample solution into ES source 105 while a second syringe isbeing flushed and cleaned prior to loading the next sample solution tobe sprayed into the second injector needle and syringe. The syringes canbe partially or completely filled with sample solution for each FIA run.The fill and spray liquid flow rates are determined by the syringe sizeused and the plunger movement rate as programmed in the autoinjector.Commercially available autoinjectors are configured to flush theinternal bore of the syringe and injection needle and wash the injectionneedle external surface.

In an alternative embodiment to the invention, injector needle 100 isconnected to a fluid transfer tube that may also serve as a samplesolution reservoir instead of syringe 108. A fluid pump is connected tothe opposite end of the fluid transfer tube controlling the samplesolution flow into and out of injector needle 100. In such anembodiment, the fluid pump may or may not translate with the injectorneedle. Commercially available autoinjectors like the Hewlett Packard1100 are configured with an injector needle connected to a tube made ofpeek, stainless steel or other material and the sample solution isloaded in the reverse flow direction through the injector needle andinto such tube prior to injection of the sample solution in the forwardflow direction through the injector needle tip. The sample solutionvolume removed from the sample vials and flow rate of the samplesolution loading and spraying can be programmably controlled incommercially available autoinjectors configured with transfer tubingconnected to the injector needle. Other commercially availableautoinjectors are configured with a variations of injector needle andattached solution reservoir designs. Autoinjectors can be customdesigned or modified to accommodate the translation and orientation ofloaded injector needles into ES probe assembly bore 130. Suchautoinjectors require an injector needle with sufficient rigidity topenetrate the septa of a sample vial and to push past seal 127 in ESprobe assembly 101. Sample solution 122 is removed from sample vial 123with reverse flow through injector needle 100 and held in solutionreservoir 124. Loaded injector needle 100 is moved from sample vial 123,translated to ES source probe 101 and slid through bore 130 with tip 113location programmably positioned inside, even with or slightly past tip102 depending on the application and spraying conditions desired. Samplesolution is then sprayed from tip 113 with or without pneumaticnebulization assist into ES chamber 103. A portion of the ions that areproduced in ES source 105 are directed into vacuum where they are massto charge analyzed. Variations of autoinjector designs may be configuredwhich include the apparatus and methods of the invention. In theembodiment of the invention diagrammed in FIGS. 1 and 2, injector needle100 and syringe 108 are configured as the only sample solution transportand delivery means. In this embodiment of the invention, sample crosscontamination from one FIA sample to the next is restricted to injectorneedle needle 100 and syringe or storage reservoir 108. Consequently,internal flushing of injector needle 100 and syringe or storagereservoir 108 and external flushing of injector needle 100 minimizes oreliminates cross sample contamination in FIA. Injection needle andsolution reservoir cleaning or flushing apparatus and programmablemethods are available on most commercial autoinjectors.

As shown in the embodiment of the invention diagrammed in FIG. 1, thecenterline of ES probe assembly 101 is oriented orthogonal to the boreof capillary 104. The position of probe tip 102 is located a distance r₁from the capillary centerline and a distance Z₁ from the face ofnosepiece 112. Injector needle 100 and ES probe assembly 101 can beoperated at ground potential during Electrospraying when an ES source isconfigured with dielectric capillary tube 104. The potential of an ionbeing swept through the bore of dielectric capillary tube 104 intovacuum is described in U.S. Pat. No. 4,542,293. In alternativeembodiments of the invention, the dielectric capillary can be replacedwith a nozzle or conductive (metal) capillary and ES sources can beconfigured with or without heated counter current drying gas 110. Whenconductive capillaries or orifices are configured in ES source 105,injector needle 100 and ES probe tip 102 will be operated at highelectrical potential relative to counterelectrodes 115, 112 and 114.Power supply 131 can be connected to injector needle 100 at contact 125and to ES probe assembly 101 to apply the required high electricalpotential, typically 3,000 to 6,000 volts during Electrospray operation.The Electrospray chamber 103 may be configured shorter in length andsmaller in diameter due to the reduced flow rate range and totalsolution volume sprayed in FIA applications.

ES probe assembly 101 may be integrated into the walls of Electrospraychamber 103 or may be configured to penetrate through the wall of ESchamber 103. ES probe assembly 101 may be configured with alternativeseals from that shown as seal 127 in FIG. 2D. Seal 127 in FIG. 2 may bereplaced by ferrule 132 and seal 133 of ES probe assembly 135 or byseptum seal 134 in ES probe assembly 136 as diagrammed in FIG. 3. Seals127, 132 and 136 prevent nebulizer gas flow from exiting throughentrance port 111 during Electrospraying with pneumatic nebulizationassist. O-ring seal 127 may not close when injector needle 100 isremoved from ES probe assembly 101. If nebulizing gas flow remains onwhen injector needle 100 has been removed, gas can flow out port 111 aswell as through tip 102. Flushing nebulizer gas flow through ES probeassembly 101 during the time period between each sample solutionspraying may be desirable to evaporate any solvent deposited in bore 130when injector needle 100 is removed. Alternatively, nebulizing gas flowand even ES electrical potentials may be turned off when injector needle100 is removed from ES probe assembly 101. Nebulizing gas and ESelectrical potentials can be turned on when injector needle 100 isinserted into ES probe assembly 101. Commercial autoinjectors can beprogrammed to delay the dispensing of liquid from injector needle 100for a period of time after injector needle 100 is inserted into ES probeassembly 101. This programmed delay allows the MS data acquisitionsystem time to start, the nebulizing gas flow to stabilize, and the ESlens voltages to stabilize before the sample solution is Electrosprayed.Autoinjectors can also be programmed to dispense the sample at variableflow rates to optimize FIA performance. Ferrule 132 with seal 133captured in ES probe assembly 135 of FIG. 3 may be configured to provideadequate gas sealing while imposing minimum resistance when inserting orremoving injector needle 100. Septum seal 134 mounted in ES probeassembly 136 reseals when injector needle 100 is removed. Additionalforce may be required to reinsert injector needle 100 into bore 138 ofES probe assembly 136 when compared with the force required to insertinjector needle 100 through seals 127 and 133. With alternative seals133 or 136, nebulizer gas and ES electrical potentials can remain on atall times during FIA injection or they may be turned on only wheninjection needle 100 is inserted into an ES probe assembly. Turning offnebulizing gas flow 128 reduces gas consumption between each sample FIA.

Bore 130 of ES probe assembly 101 as diagrammed in FIG. 1 is positionedorthogonal to the bore of capillary 104. Commercially available ESsources include off axis and on axis ES probes with the angles of ESprobe assembly centerlines ranging from zero to ninety degrees. U.S.Pat. No. 5,495,108 even describes ES probe positions with angles greaterthan 90 degrees. FIG. 4 shows a diagram of an embodiment of theinvention in which the bore of ES probe assembly 301 is positioned onthe same axis as the bore of capillary 304. The distance from ES probetip 312 to nosepiece 309 is set at Z₂ in FIG. 4. The value for Z₂ rangestypically from 0.5 to 2 cm for ES flow rates ranging from 0.2 to 200ul/min. ES source chamber 305 is configured with heated drying gas flow310. ES probe assembly 301 is configured with inlet port 311, nebulizergas port 307 and exit end 302 with tip 312. Injector needle 300 withattached reservoir 308 is inserted into ES probe assembly 305 asdescribed in FIGS. 1 and 2 for the orthogonal ES probe embodiment.Higher sensitivity can be achieved for lower solution flow rates usingon-axis ES probes compared with off axis probes. ES probe assemblies 101and/or 301 can be oriented at any angle relative to the capillary boreaxis. Multiple ES probe assemblies configured according to the inventioncan be mounted in the same ES chamber. ES probes configured according tothe invention can be mounted with standard ES probes in the same ESsource. Multiple ES and APCI probe assemblies configured in an APIsource are described in U.S. patent application Ser. No. 60/076,118 andincorporated herein by reference. Autoinjectors can be configured toposition injector needles in the orientation required for insertion intoES probe assemblies configured according to the invention that areoriented at various angles in an ES chamber.

An alternative embodiment to the invention is diagrammed in FIG. 5wherein injector needle 200 attached to reservoir 208 is configured todeliver sample solution through APCI nebulizer probe assembly 201. APCIsource assembly 218 comprises two APCI inlet probe assemblies 201 and213, droplet separator ball 212 in droplet transfer assembly 203,vaporizer heater 214, corona discharge needle 215 nosepiece 216,capillary 204, heated counter current gas 210 and cylindrical lens 217in APCI chamber 205. Droplet separator ball 212 may or may not beincluded depending on the liquid flow rates being sprayed from APIprobes 201 and/or 213. Bore 220 of APCI nebulizer probe assembly 201 isoriented orthogonal to the APCI source centerline defined by theextension of the capillary orifice 209 centerline. The centerline of asecond APCI nebulizer probe 213 which connects to a liquid transferline, is positioned along the APCI source centerline. Sample solutionsdelivered through injector needle 200 and APCI probe assembly 213 aresprayed into chamber 222 before being swept through vaporizer 214.Sample solutions may be sprayed simultaneously or sequentially from APCInebulizer probe assemblies 201 and 213. Sprayed liquid droplets fromAPCI probe assembly 201 or 213 evaporate as they pass through vaporizerheater 214. Kilovolt electrical potentials are applied to coronadischarge needle 215 to form a corona discharge near the exit end ofvaporizer heater 214. Sample bearing vapor is ionized as it passedthrough the corona discharge region near the tip of needle 215. Aportion of the ions formed in the corona discharge region are directedagainst counter current gas flow 210 toward capillary orifice 209 by theelectric fields formed from the electrical potentials applied tocylindrical lens 217 and needle 215, nosepiece 216 and capillaryentrance lens 223. Ions are swept into vacuum through the bore ofcapillary 204 where they are mass to charge analyzed.

In an alternative embodiment of the invention, FIG. 6 shows a diagram ofAPCI nebulizer probe 401 configured similar to APCI probe 201. Injectorneedle 400 attached to solution reservoir 408 can be slid throughadjustable needle port 411, seal 413 and bore 412 of APCI nebulizerprobe assembly 401. Nebulizer gas flow enters port 407 and flows betweenthe inner diameter of bore 412 and the outer diameter of insertedinjector needle 400. The tip of the inserted injector needle 400 ispositioned relative to exit orifice 202 and 402 of APCI probe assemblies201 or 401 respectively to optimize the nebulization efficiency of theliquid spray. The tip of injector needle 400 may be positioned slightlyinside, even with or extended beyond exit orifice 202 or 402 dependingon liquid and nebulizer gas flow rates. Sample solution sprayed frominjector needle 400 passes through the droplet transfer region 403,through vaporizer heater 414 and into APCI chamber 405. Ions formed fromthe vaporized sample solution in the corona discharge region aredirected against counter current gas flow 410 into orifice 409 ofcapillary 404 by the electric fields in APCI source chamber 405. Ionsentering capillary orifice 409 are swept into vacuum through the bore tocapillary 404 where they are mass to charge analyzed with a massspectrometer.

The sequence of sample solution pick up and transfer to an API probeshown in FIG. 2 can be applied to injector needles 200 and 400 and APCInebulizer probe assemblies 201 and 401. As in the Electrospray ionsource embodiment, an APCI source can be operated in flow injectionanalysis mode using apparatus and methods according to the invention.Injector valves, transfer tubing, fluid line connections, probe liquidtransfer tubing and additional fluid flow pumps can be eliminated whenan injector needle and sample solution reservoir is configured andoperated according to the invention in APCI MS FIA. Alternative sealsmay be configured in APCI probe assemblies 201 or 401 as diagrammed inFIG. 3 for ES probe assemblies 135 and 136. Bore 220 of APCI nebulizerprobe assembly 201 may be oriented at any angle relative to the APCIsource centerline. Multiple APCI nebulizer probes configured accordingto the invention may be mounted to droplet transfer assembly 203. As inthe ES probe assembly embodiment of the invention, removable injectorneedle 200 or 400 may be attached to a syringe, a solution reservoir ora liquid transfer tube. Such a liquid transfer tube may be connected toa fluid flow pump to deliver sample solution through injector needle200. Sample solution may be introduced in this manner to deliver acalibration solution into the ES or APCI sources sequentially orsimultaneously with a second sample solution spray as is described inU.S. patent application Ser. No. 60/076,118. The movement andpositioning of injector needle 200 and attached solution reservoir 208may be controlled manually or using a programmable mechanical apparatussuch as an autoinjector. Autoinjectors can be configured to insertinjector needle 208 into bore 220 of APCI probe assembly 201 at anyangle required by the APCI source probe geometry. The total samplesolution volume loaded and the liquid flow rates passing throughinjector needle 200 can be programmably controlled by an autoinjectorapparatus. Multiple solution samples can be run sequentially in an ESand/or APCI source delivered from an autoinjector containing multiplesample vials from which sample solution can be loaded into an injectorneedle configured according to the invention.

It is obvious to one skilled in the art that variations of embodimentsof the invention may include but are not limited to combinations of:

-   -   1. The ES or APCI probe assembly centerline to vacuum orifice        centerline angles (f) may range from 0° to 180°;    -   2. ES or APCI probe assembly tip locations (r₁, z₁,) where r_(i)        may equal any distance, and z_(i) may equal any distance within        an ES chamber;    -   3. An API source capillary or orifice mounted in a position with        its centerline ranging from horizontal to vertical;    -   4. The ES or APCI probe assembly can be combined with other ES        probe types or APCI probes in the same API source as described        in U.S. patent application Ser. No. 60/076,118;    -   5. The ES probe assembly may include pneumatic assisted        Electrospray nebulization, ultrasonic assisted Electrospray        nebulization or other nebulizer type or may be configured for        unassisted Electrospray operation;    -   6. Electrospray probe may include two, three or more layer        construction for use with or without additional liquid layer        flow;    -   7. Multiple injector needles directed to one ES or APCI probe        assembly or multiple injector needles directed to multiple ES or        APCI probe assemblies; and    -   8. Multiple ES or APCI probe assemblies configured according to        the invention and mounted in one API source apparatus.

An alternative embodiment of the reusable injector needle is shown inFIG. 7, wherein the internal bore of injector needle 420 is filled withpacking material 421. Packing material 421 may comprise but is notlimited to coated beads similar to those used in liquid chromatographycolumns or filter material. Frits 422 and 423 are configured at each endof the bore of injector needle 420 to retain packing material 421 duringloading and delivery of sample solution through injector needle 420.FIG. 7A shows an external view of injector needle 420 attached tosyringe 424 shown in cross section with plunger 425 and plunger seal 426located in the full forward or empty position. FIG. 7B is a diagram ofthe cross section of packed injector needle 420 and syringe 424 prior toloading solvent and sample solution. The inclusion of packing materialin injector needle 420 allows desalting and other sample cleanup as wellas separation of components in the sample solution during API MS FIA. Assample solution is loaded from the sample vial, it passes through thepacking material in injector needle 420. As an example, when FIA ofpeptides is performed, the packing material may be selected to be C18coated beads. This C18 packing material is similar to the media packedin high pressure liquid chromatography columns that are used to conductgradient separations of peptides. A partial sample component gradientseparation can be achieved in FIA when the sample solution is deliveredto an API source in through the packed injector needle 420 connected toa syringe loaded with a solvent gradient as shown in FIG. 7C.

Referring to FIG. 7C, syringe 424 with injector needle 420 is loadedwith a series of solutions of different compositions 428 through 433from sequentially from different containers to form a solvent gradientalong the length of syringe reservoir 436. For example the compositionof solution volume 428 may be 10% water and 90% acetonitrile with 0.05%TFA, solution volume 429, 30% water and 70% acetonitrile with 0.05% TFAto solution volume 433 which may be 90% water and 10% acetonitrile with0.05% TFA. A solvent gradient is formed in syringe reservoir 436 fromloading partial syringe volumes consecutively from a series of vialscontaining solution compositions ranging, in this example from 10% to90% acetonitrile with 0.05% TFA. To divide the solvent gradient from thesample solution, air bubble volume 434 can be drawn into syringe 424before loading sample solution volume 435. The sample solutioncontaining a high percentage of water passes through the C18 mediapacked in the bore of injector needle 420 during loading. Salts andother contaminants pass through injector needle 420 into internal volume436 of syringe 424 during loading of the sample solution while thepeptide sample components of interest adsorb or stick to available siteson the C18 media in injector needle 420. When syringe 424 is loaded, itis moved and aligned with the bore of an API probe assembly and theinjector needle is introduced through the API probe bore according tothe invention as diagrammed in FIGS. 1 and 2 above. As syringe plunger425 is moved forward, sample solution solvents, salts and contaminantscontained in volume 435 are sprayed into the API source chamber whilethe peptide sample components remain adsorbed to packing material 421.Air bubble 434 and solvent gradient volume 433 through 428 pass throughinjector needle 420 as syringe 424 is emptied during ES or APCI MS FIA.As solvent volumes 433 through 428 pass through injector needle media421, the adsorbed peptides will release from the C18 packing materialwith increasing organic content of the solvent solution. Some peptidecomponent separation may occur as syringe 424 is emptied becausedifferent peptides may release from the C18 packing material atdifferent organic solvent concentrations. Peptides spraying from packedinjector needle 420 into an API source may be separated from salts orother contaminants contained in the original sample solution using thismethod. Some degree of chromatographic separation may also be achievedusing this method during API MS flow injection analysis, improving thequality of acquired MS data. Flushing or cleaning between sample runs islimited to flushing packed injector needle 420 and the inner bore ofsyringe 424 and washing the outside surface of injector needle 420.

In alternative embodiments of the invention, a fluid transfer line orother solution reservoir may replace syringe 424 in FIG. 7. The loading,emptying and positioning of packed injector needle 420 with syringe 424may be controlled manually or mechanically with programmable control.Syringe 420 configured with packed injector needle 420 may be mounted inan autoinjector which also contains several sample vials required toconduct the method described above. Packing material may extend into asolution reservoir in alternative embodiments of the invention. U.S.Pat. No. 5,572,023 describes the filling of fixed position Electrosprayneedles with chromatography packing material. In the apparatus describedin U.S. Pat. No. 5,572,023, the packed Electrospray needle is connectedto sample injector valves, transfer lines and multiple solvent pumps forconducting sample desalting of gradient separations in a single liquidflow direction. Sample cleanup and chromatographic separation in FIA canbe achieved with the apparatus configured according to the inventionwithout injector valves, liquid transfer lines or additional liquid flowpumps. According to the invention, solution flow moves through theinjector needle in the reverse direction during loading and in theforward direction during spraying. The loading of solvents and/or samplesolution through the injector needle packing material may be aided bypressurizing the solution vials or containers.

The sample solution can alternatively be loaded without including bubble434 if it is desirable to have a continuous liquid column when loadingor spraying. A continuous liquid column provides increased tensilestrength when pulling sample solution from a vial through packingmaterial. When desalting is desired without chromatographic separation,a two part solvent front may be preloaded in syringe 424 instead of anumber of different organic concentrations. In a stepwise manner, thefirst high percentage aqueous solvent volume will flush out salts andother contaminants through injector needle 420 while spraying and thesecond high percentage organic solvent volume passing through packingmaterial 421 will release the adsorbed peptide components simultaneouslyduring API source spraying. Injector needle 420 may be packed withfilter media instead of chromatography packing material to removecontaminants from the sample solution prior to sample injection into theAPI source.

In an alternative embodiment of the invention, the reusable injectionneedle is replaced by a removable or disposable injection needle. Theremovable injection needle may be comprised of a dielectric materialsuch as molded plastic, a combination of dielectric and conductivematerial or entirely of conductive material. FIG. 8 is a diagram of fourembodiments of removable or disposable injector needles configuredaccording to the invention. Removable injector needle 444 is fabricatedfrom dielectric material such as molded polyethylene. Removable injectorneedle 444 comprises the narrower needle portion 445 and the larger,diameter upper portion 446. A portion of the removable injector needleinner volume may be filled with media such as filter or chromatographypacking material. Such packing material can be used to removecontaminants from the sample solution or separate sample components asdescribed for the reusable needle embodiment. Removable injector needle440 in FIG. 8 is shown with packing material 442 filling the narrowerneedle portion of removable injector needle 440. In the embodimentshown, packing material is prevented from moving out of the bore ofremovable injector needle 440 by frits 441 an 443. Dielectric removableinjector needles 440 and 444 may be used to spray samples into APCIsources where the formation of charged liquid droplets is not required.A conductive tip is preferred for Electrospraying from a removableinjector needle. Two alternative embodiments of removable injectorneedles 448 and 454 with conductive elements configured in each tip arediagrammed in FIG. 8. In one embodiment, conductive strip 452 beginningat tip 450 and extending to the opposite opening end 451 is molded intoor attached to the inside surface of removable injector needle 448.Conductive strip 452 provides electrical contact with the samplesolution at tip 450 and a contact point at end 451 duringElectrospraying. An alternative means to provide an electrical contactwith the Electrospraying solution is shown with removable injectionneedle 454. Conductive material 455 is attached to the external surfaceof removable injection needle 454 surrounding tip 456. Conductivematerial 455 serves to provide electrical contact between the solutionspraying from tip 456 with an electrical conductor external to removableinjector needle 454 during Electrospray operation.

FIG. 9 diagrams embodiments of the invention wherein removable injectionneedles are used to load sample solution and conduct API MS flowinjection analysis. Referring to FIG. 9, pipette device 470 is movedinto position and inserted into removable injector needle 474 in set473. Contact between the pipette device taper end 497 and removableinjector needle 474 at point 475 provides a seal and sufficient frictionto allow temporary attachment of removable injector needle 474 to taperend 497 of pipette device 470. Plunger 471 of pipette device 470 islocated in its forward position in FIG. 9A when picking up removableinjector needle 474. Channel 472 in pipette device 470 connects theplunger chamber volume with the internal volume of the attached injectorneedle 474. Removable injector needle 474, configured with electricalconductor 476 surrounding tip 498, is moved from set 473 to vial 480containing sample solution 479 as shown in FIG. 9B. Plunger 471 is movedin the reverse direction to load sample solution 478 into removableinjection needle 474 through the opening at tip 498. Volume 477 createdby retracting plunger 471 determines the volume of sample solutionloaded into removable injector needle 474. The desired sample solutionvolume 481 is loaded into removable injector tip 474, and if necessaryfor greater volume, into plunger volume 477. Pipette device 470 withloaded attached injector needle 474 are moved from container 480 to aposition where tip 498 can be inserted into to the bore of API probeassembly 482 as shown in FIG. 9C and D. When removable injector needle474 is fully inserted into API probe assembly 482, the outer diameter ofremovable injector needle 474 forms a gas seal with probe seal 483.Electrical contact to ground or power supply 494 is made to conductivematerial 476 through contact 484 and connection 488. Nebulizer gas flow487 enters API probe assembly 482 through port 468 and exits at probetip 499 flowing through the annulus between conductive material 476 andthe bore of API probe 482. The forward movement of plunger 471 pushessample solution 481 out through tip 498 creating ES or APCI spray 486.

An alternative embodiment of API probe assembly 489 is shown in FIG. 9E.A gas seal is formed by contact point 491 between the API probe assemblybody and removable injector needle 474. Electrical contact is madedirectly between conductive material 476 and the conductive body of APIprobe assembly 489 due to the taper of tip 489 mating with API probeassembly inner bore 469. API probe assembly 489 may be grounded orconnected to voltage supply 493 for ES sources where it is required thatthe ES probe be operated at kilovolt potentials. Nebulizer gas 490enters API probe assembly 489 through port 466 and exits at API probetip 465 where nebulizer gas flow 495 aids in forming spray 496 fromsample solution in ES and APCI source operation. Typically, tip 498 ofremovable injection needle 474 has a small diameter and consequently isnot sufficiently rigid to push through a seal or a septum. Entrance end467 of API probe assembly 489 is tapered to guide the thin filament tubeend 498 through inner bore 469. A manual injector or autoinjector may beconfigured with a separate rigid tube that is use to pierce a septumsealing a sample solution vial. The removable injection needle can thenbe passed through the rigid tube passing through the septum to loadsample solution from the sample vial. After flow injection analysis iscompleted, pipette device 470 with attached injector needle 474 iswithdrawn from API probe assembly 489 or 482. Removable injector needle474 is detached from pipette device 470, a new removable injector needleis attached to pipette device 470 and the FIA cycle as diagrammed inFIG. 9 is repeated. No wash cycle is required when removable injectorneedles are used in flow injection analysis resulting in decreased cycletime per sample. Total sample throughput can be increased when usingremovable injector needles in FIA without any sample carryover orcontamination from sample to sample.

Filling, emptying and positioning of pipette device 470 can becontrolled manually or using programmable mechanical devices such asautoinjectors or automatic pipette controllers. Alternative embodimentsof the invention can be configured to control the filling, emptying andpositioning of removable injection needles as is diagrammed in FIG. 10.Referring to FIG. 10A, syringe 540 with attached needle 553 wheninserted into removable injector needle 544 forms a seal a point 547 andelectrical contact with inner conducting strip 551. Removable injectorneedle 544 is shown with tip end 555 filled with packing material 548contained by frits 549 and 550. Electrical contact between ground orvoltage supply 557 and tip 556 is made through conducting strip 545,needle 553 and electrical contact 552 when tip 556 is operated as anElectrospray tip. Reverse and forward movement of plunger 541 fills orempties reservoir 542 with sample solvent or sample solutionrespectively through removable injector needle 544 during API MS FIA. Analternative embodiment of the invention is shown in FIG. 10B. Removableinjector needle 560 is configured with conductive material 565 coatingthe outer surface of packed tip 562. Electrical connection with tip 567to ground or voltage supply 563 is made through contact betweenconductive material 565 and an API probe assembly as was diagrammed inFIG. 9. No direct electrical connection is made between tip 567 andsyringe needle 568 or syringe 561 in the embodiment diagrammed in FIG.10B eliminating any electrical connections between syringe 561 and avoltage supply or ground potential. An alternative embodiment of theinvention is diagrammed in FIG. 10C wherein removable injector needle570 with electrically conductive strip 571 makes an electricalconnection to ground or voltage supply 575 through electrical contactwith the conductive tapered portion 573 of pipette device 574.Variations and/or combinations of embodiments shown may be configuredand used according to the invention to perform flow injection analysiswith atmospheric pressure ion sources interfaced to mass spectrometers,ion mobility analyzers or other analytical devices.

FIG. 11 shows the results acquired from ES MS flow injection analysisusing the embodiment of the invention diagrammed in FIGS. 1 and 2. FIG.11 includes a total ion current TIC) trace in curve 500 and five massspectra 501-505 acquired by spraying sample solution into anElectrospray ion source configured as diagrammed in FIG. 1. A 100 ulsyringe mounted in a Leap HTS PAL autoinjector was used to load anddeliver sample solution to the ES probe assembly. The autoinjector flowrate when spraying the sample solution from the injector needle attachedto the syringe was set at 200 ul/min for the data acquired in FIG. 11.Three injections each were made of five different samples as are shownin TIC trace 500 of FIG. 11. The first three TIC peaks 506 are ofTri-Tyrosine injected at a concentration of 50 pmole/μL. Mass spectrum501 was acquired under one of the TIC peaks of 506. Mass spectrum 501shows the singly charged protonated molecular ion peak 511 ofTri-Tyrosine peak which hs a measured mass to charge value of 508.Injections of 10 pmole/ul sample solutions of protein cytochrome-C formthe second three TIC peaks 507 corresponding to injections four throughsix. Mass spectrum 502 of cytochrome-C was acquired under one of the TICpeaks in 507. Mass spectrum 502 shows peaks 512, 513, 514, 515, and 516of multiply charged protonated molecular ions of cytochrome-Ccorresponding to mass to charge values 688, 728, 773, 825, and 884respectfully. The third set of TIC peaks 508 were acquired by injectingsample solution containing 1 pmole/ul of gramicidin-S. Mass spectrum 503acquired under one of these injection peaks 503, shows the doublycharged protonated molecular ion peak 517 of gramicidin-S. The fourthset of three TIC peaks 509 were acquired by injecting a sample solutioncontaining 11 pmole/ul of Lincomycin. Mass spectrum 504 acquired underone of the TIC peaks 509, shows the singly charged protonated molecularion peak 518 of Lincomycin with a measured mass to charge of 407. Thelast three TIC peaks 510 were acquired by sequentially injecting samplesolutions containing 82 pmole/ul of reserpine. Mass spectrum 505 wasacquired under one of the TIC peaks 510 and shows the singly chargedprotonated molecular ion peak 519 of reserpine which has a measured massto charge value of 609. The data shown in FIG. 11 is an example of ES MSFIA acquired according to the invention with no injector valve, transferlines, probe transfer volumes, tubing connectors or additional fluidflow pumps. No cross talk or sample carryover is observed in TIC trace500 or acquired mass spectra 501-505.

Having described this invention with regard to specific embodiments, itis to be understood that the description is not meant as a limitationsince further modifications and variations may be apparent or maysuggest themselves. It is intended that the present application coverall such modifications and variations, including those as fall withinthe scope of the appended claims.

References Cited

The following references are referred to above, the disclosures of whichare hereby fully incorporated herein by reference:

U.S. Patent Documents:

5,495,108 Feb. 27, 1996, Apffel, James; Werlich, Mark; Bertach, James.Provisional Sep. 12, 1997, Adrien Jr., Bruce A, Sansone, Michael A,Whitehouse, Craig M. 4,542,293 Sep. 17, 1985, Fenn, John B., Yamashita,Masamichi, Whitehouse, Craig. 5,572,023 Nov. 5, 1996, Caprioli, Richard.4,531,056 Jul. 23, 1985 Labowski, Michael, Fenn John, B., Yamashita,MasamichiPublications:

T. Wang, L. Zeng, T. Strader, L. Burton, and Daniel B. Kassel,Proceedings of the 46^(th) ASMS Conference on Mass Spectrometry, 1034,1998.

1. An apparatus for producing ions from chemical species in solutioncomprising: a) a needle for withdrawing a sample bearing solution from acontainer, sold needle configured to draw said sample bearing solutiontherein and spray said sample bearing solution therefrom; b) an ionsource operated a substantially atmospheric pressure, a probe assemblyin said ion source which produces ions from sample bearing solutions,said probe assembly having an opening for removably inserting of saidneedle, said needle and probe assembly configured to produce ions fromchemical species in said sample solution as it is sprayed from theneedle with the needle inserted into the opening of the probe assembly;c) a vacuum region in fluid communication with said ion source forreceiving said ions; and d) means for mass analyzing said ions in saidvacuum region.
 2. An apparatus according to claim 1, wherein said ionsource is an Electrospray source.
 3. An apparatus according to claim 2,wherein said Electrospray source has pneumatic assist means.
 4. Anapparatus according to claim 2, wherein said Electrospray source hasultrasonic assist means.
 5. An apparatus according claim 2, wherein saidElectrospray source has nebulization assist means.
 6. An apparatusaccording to claim 2, wherein said Electrospray source comprises twotube layers.
 7. An apparatus according to claim 2, wherein saidElectrospray source comprises three or more tube layers.
 8. An apparatusaccording to claim 1, wherein said ion source is an Atmospheric PressureChemical Ionization source.
 9. An apparatus according to claim 8,wherein said Atmospheric Pressure Chemical Ionization source haspneumatic assist means.
 10. An apparatus according to claim 8, whereinsaid Atmospheric Pressure Chemical Ionization source has ultrasonicassist means.
 11. An apparatus according to claim 8, wherein saidAtmospheric Pressure Chemical Ionization source has nebulization assistmeans.
 12. An apparatus according to claim 8, wherein said AtmosphericPressure Chemical Ionization source comprises two tube layers.
 13. Anapparatus according to claim 8, wherein said Atmospheric PressureChemical Ionization source comprises three or more tube layers.
 14. Anapparatus according to claim 1, said needle is a syringe.
 15. Anapparatus according to claim 1, wherein said needle is an injectionneedle and storage reservoir from an autoinjector.
 16. An apparatusaccording to claim 15, wherein said injection needle is an injectionneedle with its needle filled with a liquid chromatography material fordesalting of the sample.
 17. An apparatus according to claim 15, whereinsaid injection needle is an injection needle with its needle filled witha liquid chromatography material and said storage reservoir is partiallyfilled with a liquid chromatography material for desalting of thesample.
 18. An apparatus according to claim 15, wherein storagereservoir is partially filled with a liquid chromatography material fordesalting of the sample.
 19. An apparatus according to claim 15, whereinsaid injection needle is an injection needle with its needle filled witha liquid chromatography material for liquid chromatography separations.20. An apparatus according to claim 15, wherein said injection needle isan injection needle filled with a liquid chromatography material andsaid storage reservoir is partially filled with a liquid chromatographymaterial for liquid chromatography separations.
 21. An apparatusaccording to claim 15, wherein said storage reservoir is partiallyfilled with a liquid chromatography material for liquid chromatographyseparations.
 22. An apparatus according to claim 15, wherein saidinjection needle is an injection needle having an inside surface alongsaid needle, wherein said inside surface is coated with a liquidchromatography material for desalting of the sample.
 23. An apparatusaccording to claim 15, wherein said injection needle is an injectionneedle having an inside surface along said needle, wherein said insidesurface is coated with a liquid chromatography material and said storagereservoir is partially coated with a liquid chromatography material fordesalting of the sample.
 24. An apparatus according to claim 15, whereinsaid storage reservoir is partially coated with a liquid chromatographymaterial for desalting of the sample.
 25. An apparatus according toclaim 15, wherein said injection needle is an injection needle having aninside surface along said needle, wherein said inside surface is coatedwith a liquid chromatography material for liquid chromatographyseparations.
 26. An apparatus according to claim 15, wherein saidinjection needle is an injection needle having an inside surface alongsaid needle, wherein said inside surface is coated with a liquidchromatography material and said storage reservoir is partially coatedwith a liquid chromatography material for liquid chromatographyseparations.
 27. An apparatus according to claim 15, wherein saidstorage reservoir is partially coated with a liquid chromatographymaterial for liquid chromatography separations.
 28. An apparatusaccording to claim 15, wherein said injection needle is an injectionneedle having an inside surface along said needle, wherein said insidesurface is coated with a liquid chromatography material and said storagereservoir is partially coated with a liquid chromatography material forliquid chromatography separations.
 29. An apparatus as set forth inclaim 1, wherein said needle comprises an electrically conductivematerial to form an electrode.
 30. An apparatus according to claim 1,wherein said means for mass analyzing said ions comprises aTime-Of-Flight mass spectrometer.
 31. An apparatus according to claim 1,wherein said means for mass analyzing said ions comprises a Quadrupolesmass spectrometer.
 32. An apparatus according to claim 1, wherein saidmeans for mass analyzing said ions comprises an Ion Trap massspectrometer.
 33. An apparatus according to claim 1, wherein said meansfor mass analyzing said ions comprises a Fourier Transform massspectrometer.
 34. An apparatus according to claim 1, wherein said meansfor mass analyzing said ions comprises a magnetic sector massspectrometer.
 35. An apparatus according to claim 1, wherein said meansfor mass analyzing said ions comprises a hybrid mess spectrometer.
 36. Amethod for producing ions from chemical species in a solutioncomprising: a) withdrawing a sample bearing solution from a vial with aneedle, b) removably inserting the sample bearing solution containingneedle into an opening in a probe assembly of an ion source, said ionsource operated at atmospheric pressure to produce ions from samplebearing solutions, c) spraying said sample bearing solution from saidneedle, said probe assembly and needle operating to produce ions as thesample bearing solution is sprayed, and d) delivering said ions into avacuum region.
 37. A method according to claim 36, wherein said ionsource is an Electrospray source.
 38. A method according to claim 37,wherein said Electrospray source has pneumatic assist means.
 39. Amethod according to claim 37, wherein said Electrospray source hasultrasonic assist means.
 40. A method according to claim 37, whereinsaid Electrospray source has nebulization assist means.
 41. A methodaccording to claim 37, wherein said Electrospray source comprises twotube layers.
 42. A method according to claim 37, wherein saidElectrospray source comprises three or more tube layers.
 43. A methodaccording to claim 36, wherein said ion source is an AtmosphericPressure Chemical Ionization source.
 44. A method according to claim 43,wherein said Atmospheric Pressure Chemical Ionization source haspneumatic assist means.
 45. A method according to claim 43, wherein saidAtmospheric Pressure Chemical Ionization source has ultrasonic assistmeans.
 46. A method according to claim 43, wherein said AtmosphericPressure Chemical Ionization source has nebulization assist means.
 47. Amethod according to claim 43, wherein said Atmospheric Pressure ChemicalIonization source comprises two tube layers.
 48. A method according toclaim 43, wherein said Atmospheric Pressure Chemical Ionization sourcecomprises three or more tube layers.
 49. A method according to claim 36,wherein said needle is a syringe.
 50. A method according to claim 36,wherein said needle is an injection needle and storage reservoir from anautoinjector.
 51. A method according to claim 50, wherein said injectionneedle is an injection needle with its needle filled with a liquidchromatography material for desalting of the sample.
 52. A methodaccording to claim 50, wherein said injection needle is an injectionneedle with its needle filled with a liquid chromatography material andsaid storage reservoir is partially filled with a liquid chromatographymaterial for desalting of the sample.
 53. A method according to claim50, wherein said storage reservoir is partially filled with a liquidchromatography material for desalting of the sample.
 54. A methodaccording to claim 50, wherein said injection needle is an injectionneedle filled with a liquid chromatography material for liquidchromatography separations.
 55. A method according to claim 50, whereinsaid injection needle is an injection needle filled with a liquidchromatography material and said storage reservoir is partially filledwith a liquid chromatography or liquid chromatography separations.
 56. Amethod according to claim 50, wherein said storage reservoir ispartially filled with a liquid chromatography material for liquidchromatography separations.
 57. A method according to claim 50, whereinsaid injection needle is an injection needle having an inside surfacealong said needle, wherein said inside surface is coated with a liquidchromatography material for desalting of the sample.
 58. A methodaccording to claim 50, wherein said injection needle is an injectionneedle having an inside surface along said needle, wherein said insidesurface is coated with a liquid chromatography material and said storagereservoir is partially coated with a liquid chromatography material fordesalting of the sample.
 59. A method according to claim 50, whereinsaid storage reservoir is partially coated with a liquid chromatographymaterial for desalting of the sample.
 60. A method according to claim50, wherein said injection needle is an injection needle having aninside surface along said needle, wherein said inside surface is coatedwith a liquid chromatography material for liquid chromatographyseparations.
 61. A method according to claim 50, wherein said injectionneedle is an injection needle having an inside surface along saidneedle, wherein said inside surface is coated with a liquidchromatography material and said storage reservoir is partially coatedwith a liquid chromatography material for liquid chromatographyseparations.
 62. A method according to claim 50, wherein said storagereservoir is partially coated with a liquid chromatography material forliquid chromatography separations.
 63. A method according to claim 50,wherein said injection needle is an injection needle having an insidesurface along said needle, wherein said inside surface is coated orfilled with a liquid chromatography material and said storage reservoiris partially coated or filed with a liquid chromatography material forliquid chromatography separations.
 64. A method as set forth in claim36, further comprising the step of creating an electrode of said needlecontaining said sample bearing solution.
 65. A method for producing ionsfrom chemical species in a solution comprising: a) withdrawing a samplebearing solution from a vial with a needle, b) removably inserting thesample bearing solution containing needle into an opening in a probeassembly of an ion source, said ion source operated at atmosphericpressure to produce ions from sample bearing solutions, c) spraying saidsample bearing solution from said needle, said probe assembly and needleoperating to produce ions as the sample bearing solution is sprayed, andd) mass analyzing said ions from the ion source.
 66. A method accordingto claim 65, wherein said ions are mass analyzed in a Time-Of-Flightmass spectrometer.
 67. A method according to claim 65, wherein said ionsare mass analyzed in a Quadrupoles mass spectrometer.
 68. A methodaccording to claim 65, wherein said ions are mass analyzed in an IonTrap mass spectrometer.
 69. A method according to claim 65, wherein saidions are mass analyzed in a Fourier Transform mass spectrometer.
 70. Amethod according to claim 65, wherein said Ions are mass analyzed in amagnetic sector mass spectrometer.
 71. A method according to claim 65,wherein said ions are mass analyzed in a hybrid mass spectrometer.
 72. Amethod according to claim 65, wherein said ion source is an Electrospraysource.
 73. A method according to claim 72, wherein said Electrospraysource has pneumatic assist means.
 74. A method according to claim 72,wherein said Electrospray source has ultrasonic assist means.
 75. Amethod according to claim 72, wherein said Electrospray source hasnebulization assist means.
 76. A method according to claim 72, whereinsaid Electrospray source comprises two tube layers.
 77. A methodaccording to claim 72, wherein said Electrospray source comprises threeor more tube layers.
 78. A method according to claim 65, wherein saidion source is an Atmospheric Pressure Chemical Ionization source.
 79. Amethod according to claim 78, wherein said Atmospheric Pressure Chemicallonization source has pneumatic assist means.
 80. A method according toclaim 78, wherein said Atmospheric Pressure Chemical Ionization sourcehas ultrasonic assist means.
 81. A method according to claim 78, whereinsaid Atmospheric Pressure Chemical Ionization source has nebulizationassist means.
 82. A method according to claim 78, said AtmosphericPressure Chemical Ionization source, comprises two tube layers.
 83. Amethod according to claim 78, wherein said Atmospheric Pressure ChemicalIonization source comprises three or more tube layers.
 84. A methodaccording to claim 65, wherein said needle is a syringe.
 85. A methodaccording to claim 65, wherein said needle is an injection needle andstorage reservoir from an autoinjector.
 86. A method according to claim85, wherein said injection needle is filled with a liquid chromatographymaterial for desalting of the sample.
 87. A method according to claim85, wherein said injection needle is filled with a liquid chromatographymaterial and said storage reservoir is partially filled with a liquidchromatography material for desalting of sample.
 88. A method accordingto claim 85, wherein said storage reservoir is partially filled with aliquid chromatography material desalting of the sample.
 89. A methodaccording to claim 85, wherein said injection needle is filled with aliquid chromatography material for liquid chromatography separations.90. A method according to claim 85, wherein said injection needle isfilled with a liquid chromatography material and said storage reservoiris partially filled with a liquid chromatography material for liquidchromatography separations.
 91. A method according to claim 85, whereinsaid storage reservoir is partially filled with a liquid chromatographymaterial for liquid chromatography separations.
 92. A method accordingto claim 85, wherein said injection needle is an injection needle havingan inside surface along said needle, wherein said inside surface iscoated with a liquid chromatography material for desalting of thesample.
 93. A method according to claim 85, wherein said injectionneedle is an injection needle having an inside surface along saidneedle, wherein said inside surface is coated with a liquidchromatography material and said storage reservoir is partial coatedwith a liquid chromatography material for desalting of the sample.
 94. Amethod according to claim 85, wherein said storage reservoir ispartially coated with a liquid chromatography material for desalting ofthe sample.
 95. A method according to claim 85, wherein said injectionneedle is an injection needle having an inside surface along saidneedle, wherein said inside surface is coated with a liquidchromatography material for liquid chromatography separations.
 96. Amethod according to claim 85, wherein said injection needle is aninjection needle having an inside surface along said needle, whereinsaid inside surface is coated with a liquid chromatography material andsaid storage reservoir is partially coated with a liquid chromatographymaterial for liquid chromatography separations.
 97. A method accordingto claim 85, wherein said storage reservoir is partially coated with aliquid chromatography material for liquid chromatography separations.98. A method according to claim 85, wherein said injection needle is aninjection needle having an inside surface along said needle, whereinsaid inside surface is coated or filled with a liquid chromatographymaterial and said storage reservoir is partially coated or filled with aliquid chromatography material for liquid chromatography separations.99. A method as set forth in claim 65, further comprising the step ofcreating an electrode of said needle containing said sample bearingsolution.
 100. A method for producing ions from chemical species in asolution comprising: a) withdrawing a sample bearing solution from avial with a needle, b) removably inserting the sample bearing solutioncontaining needle into an opening in a probe assembly of an ion source,said ion source operated at atmospheric pressure to produce ions fromsample bearing solutions, c) spraying said sample bearing solution fromsaid needle, said probe assembly and needle operating to produce ions asthe sample bearing solution is sprayed, d) delivering said ions into avacuum region; and e) mass analyzing said ions.
 101. A method accordingto claim 100, wherein said ions are mass analyzed in a Time-Of-Flightmass spectrometer.
 102. A method according to claim 100, wherein saidions are mass analyzed in a Quadrupoles mass spectrometer.
 103. A methodaccording to claim 100, wherein said ions are mass analyzed in an IonTrap mass spectrometer.
 104. A method according to claim 100, whereinsaid ions are mass analyzed in a Fourier Transform mass spectrometer.105. A method according to claim 100, wherein said ions are massanalyzed in a magnetic sector mass spectrometer.
 106. A method accordingto claim 100, wherein said ions are mass analyzed in a hybrid massspectrometer.
 107. A method according to claim 100, wherein said ionsource is an Electrospray source.
 108. A method according to claim 100,wherein said Electrospray source has pneumatic assist means.
 109. Amethod according to claim 107, wherein said Electrospray source hasultrasonic assist means.
 110. A method according to claim 107, whereinsaid Electrospray source has nebulization assist means.
 111. A methodaccording to claim 107, wherein said Electrospray source comprises twotube layers.
 112. A method according to claim 107, wherein saidElectrospray source comprises three or more tubs layers.
 113. A methodaccording to claim 100, wherein said ion source is an AtmosphericPressure Chemical Ionization source.
 114. A method according to claim113, wherein said Atmospheric Pressure Chemical Ionization source haspneumatic assist means.
 115. A method according to claim 113, whereinsaid Atmospheric Pressure Chemical Ionization source has ultrasonicassist means.
 116. A method according to claim 113, wherein saidAtmospheric Pressure Chemical Ionization source has nebulization assistmeans.
 117. A method according to claim 113, wherein said AtmosphericPressure Chemical Ionization source, comprises two be layers.
 118. Amethod according to claim 113, wherein said Atmospheric PressureChemical Ionization source comprises three or more tube layers.
 119. Amethod according to claim 100, wherein said needle is a syringe.
 120. Amethod according to claim 100, wherein said needle is an injectionneedle and storage reservoir from an autoinjector.
 121. A methodaccording to claim 120, wherein said injection needle is filled with aliquid chromatography material for desalting of the sample.
 122. Amethod according to claim 120, wherein said injection needle is filledwith a liquid chromatography material and said storage reservoirpartially filled with a liquid chromatography material for desalting ofthe sample.
 123. A method according to claim 120, wherein said storagereservoir is partially filled with a liquid chromatography material fordesalting of the sample.
 124. A method according to claim 120, whereinsaid injection needle is filled with a liquid chromatography materialfor liquid chromatography separations.
 125. A method according to claim120, wherein said injection needle is filled with a liquidchromatography material and said storage reservoir is partially filledwith a liquid chromatography material for liquid chromatographyseparations.
 126. A method according to claim 120, wherein said storagereservoir is partially filled with a liquid chromatography material forliquid chromatography separations.
 127. A method according to claim 120,wherein said injection needle is an injection needle having an insidesurface along said needle, wherein said inside surface is coated with aliquid chromatography material for desalting of the sample.
 128. Amethod according to claim 120, wherein said injection needle is aninjection needle having an inside surface along said needle, whereinsaid inside surface is coated with a liquid chromatography material andsaid storage reservoir is partial coated with a liquid chromatographymaterial for desalting of the sample.
 129. A method according to claim120, wherein said storage reservoir is partially coated with a liquidchromatography material for desalting of the sample.
 130. A methodaccording to claim 120, wherein said injection needle is an injectionneedle having an inside surface along said needle, wherein said insidesurface is coated with a liquid chromatography material for liquidchromatography separations.
 131. A method according to claim 120,wherein said injection needle is an injection needle having an insidesurface along said needle, wherein said inside surface is coated with aliquid chromatography material and said storage reservoir is partiallycoated with a liquid chromatography material for liquid chromatographyseparations.
 132. A method according to claim 120, wherein said storagereservoir is partially coated with a liquid chromatography material forliquid chromatography separations.
 133. A method according to claim 120,wherein said injection needle is an injection needle having an insidesurface along said needle, wherein said inside surface is coated orfilled with a liquid chromatography material and said storage reservoiris partially coated or filled with a liquid chromatography material forliquid chromatography separations.
 134. A method as set forth in claim100, further comprising the step of creating an electrode of said needlecontaining said sample bearing solution.
 135. A method as set forth inclaim 100, further comprising the step of centering the needlecontaining sample bearing solution within said probe assembly.